Forkhead Box Protein P3 Research Articles

Modulatory Effects of Metformin Alone and in Combination with Cimetidine and Ibuprofen on T Cell-related Parameters in a Breast Cancer Model.

Metformin, cimetidine, and ibuprofen separately exhibit immunomodulatory and anti-tumorigenic effects. Herein, the impacts of metformin alone and in combination with cimetidine/ibuprofen on some Th1- and regulatory T (Treg) cell-related parameters were evaluated using a breast cancer (BC) model. For establishing the BC model, four groups of Balb/c mice were challenged with the carcinoma cell line. After 11-30 days post-induction, they were treated intraperitoneally (with metformin (200 mg/kg), "metformin plus cimetidine (20 mg/kg)"; "metformin plus ibuprofen (20 mg/kg)", or with all three drugs in mentioned doses. Untreated BC and without tumor mice were enrolled as control groups. On day 31, splenic Th1 and Treg cell frequencies, serum interferon-gamma (IFN-γ), and transforming growth factor-beta (TGF-β) concentration, and intra-tumoral T-bet, TGF-β, and forkhead box protein P3 (FOXP3) expression were measured; using flow cytometry, enzyme-linked immunosorbent assay (ELISA), and real-time-PCR, respectively. Treatment of the BC mice with metformin alone and in combination with cimetidine and/or ibuprofen enhanced the frequency of Th1 cells, and IFN-γ concentration, while it resulted in a decrease in the frequency of Treg cells, serum TGF-β concentration, and the expression of FOXP3 and TGF-β compared with un-treated BC mice. FOXP3 expression in the metformin-treated group was lower in mice who received combination therapy. Survival rate and body weight were increased, while tumor size and spleen index were reduced in mice treated with metformin alone and its combination with cimetidine and/or ibuprofen. No remarkable differences were found between metformin-treated mice and those who received combination therapies regarding Th1 and Treg cell percentages, TGF-β expression, body weight, tumor size, and spleen index. The benefits of combinational therapy may be largely attributed to metformin. Immunotherapeutic potentials of metformin in cancers need further considerations.

Nanocurcumin supplementation ameliorates Behcet’s disease by modulating regulatory T cells: A randomized, double‐blind, placebo‐controlled trial

Current research was designed to assess the effects of nanocurcumin supplementation on regulatory T (Treg) cells frequency and function in Behçet's disease (BD). In this randomized double-masked, placebo-controlled trial, 36 BD subjects were randomly put into two groups to take one 80 mg nanocurcumin capsule or placebo daily for 8 weeks. Before and after trial, disease activity, Treg cells frequency and expression of related immunologic parameters including forkhead box protein P3 (Foxp3) transcription factor messenger RNA (mRNA) and microRNAs (miRNAs) such as miRNA-25 and miRNA-106b as well as cytokines including transforming growth factor (TGF)-β and interleukin (IL)-10 were studied. Thirty-two patients (17 in the nanocurcumin and 15 in the placebo groups) completed the trial. Treg cells frequency increased significantly in the nanocurcumin group compared with baseline (P < 0.001) and placebo group (P < 0.001). Moreover, FoxP3, TGF-β, IL‐10, miRNA-25, and miRNA-106b mRNA expression levels increased considerably in the nanocurcumin group compared to baseline (P < 0.001) and placebo group (P < 0.001, P < 0.001, P = 0.025, P = 0.011, and P < 0.001, respectively). Significant increases in serum TGF-β and IL-10 were seen in nanocurcumin group compared with baseline (P < 0.001) and placebo group (P = 0.001 and P < 0.001, respectively). Significant decrease in disease activity was found in nanocurcumin group compared with placebo group (P = 0.044). Our study provided a promising view for desirable effects of nanocurcumin supplementation in improving immunological parameters and disease activity in BD.

CD4+ CD25+ regulatory T-cells role in tumor microenvironment of the squamous cell carcinoma.

Introduction: Squamous cell carcinoma (SCC) is the most common skin cancer with a high rate of death. Different lymphocyte populations play an important role in modulating the immune response in the tumor microenvironment. The increase in the proportion of cluster of differentiation (CD)4+ CD25+ regulatory T-cell (Treg) lymphocytes is associated, in different studies, with the increase of the cell multiplication rate. Aim: To analyze the Treg lymphocyte subpopulations and to correlate the results with the presence of the CD8+ cytotoxic T-cell (Tc) lymphocyte population. Materials and Methods: Sixty primary skin SCC specimens were incubated with anti-CD8 (clone SP57) rabbit monoclonal antibody and anti-CD25 (clone 4C9) mouse monoclonal antibody. Results: The ratio of the intratumoral/peritumoral CD4+ CD25+ forkhead box protein p3 (Foxp3) lymphocytes was 0.46, emphasizing that at tumor margins, where tumor aggressiveness is higher, these lymphocytes subpopulations facilitate tumor progression. The comparative analysis of the tumor microenvironment profile revealed that in the case of intratumoral immune response, the number of Tc-type lymphocytes (CD8+) was 3.34 times higher compared to Treg lymphocytes (p<0001). In the peritumoral area, the number of Tc lymphocytes was 5.05 times higher compared to Treg lymphocytes (p<0001). Conclusions: Treg lymphocytes inhibition may cause the suppression of the antitumoral cell immune response in the tumor environment. We believe that Treg lymphocytes should represent a focus of interest for a new personalized therapy. New studies are needed to better understand the immune response in the tumor microenvironment.

Are Serum Levels of Nuclear Factor Kappa B and Forkhead Box Protein P3 in Patients with Non-Alcoholic Fatty Liver Disease Related to Severity of Fibrosis?

BACKGROUND Inflammation has a significant impact on the development and progression of fatty liver diseases.In this study, we aimed to investigate the relation between serum levels of nuclear factor kappa B (NFkB) and Forkhead box protein P3 (FOXP3)with fibrosis severity among patients with non-alcoholic fatty liver disease(NAFLD). METHODS In a prospective study, the patients suspicios of havingfatty liver were enrolled. The exclusion criteria lack of viral hepatitis, autoimmune hepatitis, Wilson's or other known liver diseases,history of liver or biliary surgery,bariatric surgery, and medications that influence liver metabolism. The participantsunderwent liver fibroscan. According to liver fibrosis, the patients weredivided into two groups; 1)fibrosis less than 7.2 KP,2)advanced NAFLD, fibrosis ≥7.3 KP. A10 cc fasting blood sample was taken from each patient for laboratory assessments.The variables between the two groups were compared using Chi-square or Fisher's exact test.The independence of cytokines was assessed by a logistic regression test. RESULTS Totally 90 patients were enrolled.The mean age was 42.21 ± 11 years. Of them, 50 and 47 participants were allocated to groups 1 and 2, respectively. In the univariate analysis, we revealed asignificant difference between age, body mass index (BMI), fasting blood glucose, liver enzymes, total cholesterol, andtriglyceride levels. Also, there was a significant difference betweenthe levels of NFKB and FOXP3 in group one compared with group two of the participants,as FOXP3(9.17 ± 10.0 vs. 18.63 ± 12.9; p < 0.001) and NFKB (1.70 ± 1.70; p < 0.01). After excluding the confounding factors, we observed a significant association between fibrosis level and cytokine levels in logistic regression. CONCLUSION Serum levels of NFKB and FOXP3 increased by advancing liver fibrosis in patients with NAFLD.This is an independent association. The identification of intermediary regulatory factors would be necessary.

Prognostic impact of tumor-infiltrating lymphocytes in non-small cell lung carcinomas.

Tumor-infiltrating lymphocytes represent a pivotal component of the host anti-tumor response. Thus, they considerably influence the evolution of cancers including non-small cell lung carcinomas. Even if, this important role is consensual, many discordant results are published in the literature about the prognostic role of the different populations of tumor-infiltrating lymphocytes. The aim of our work was to evaluate the prognostic impact of CD8+, CD4+, and forkhead box protein P3+ lymphocytes in the tumor microenvironment of non-small cell lung carcinomas. We conducted a retrospective descriptive study, which included non-small cell lung carcinomas diagnosed in the department of pathology and followed in the medical oncology department of the same hospital between 2011 and 2015. Tumor-infiltrating lymphocytes were analyzed by the immunohistochemical method for forkhead box protein P3, CD4, and CD8. Intratumoral and stromal-labeled lymphocytes were quantified by manual counting at high magnification (×400). Forkhead box protein P3+/CD8+, forkhead box protein P3+/CD4+, and CD8+/CD4+ ratios were subsequently calculated. The prognostic value of tumor-infiltrating lymphocytes was assessed in respect of overall survival, recurrence-free survival, and relapse-free survival. Thirty-nine patients were included. The mean age of patients was 59.6 years. A complete surgical resection (p = 0.009), and a CD8/CD4 ratio (p = 0.008) were prognostic factors for overall survival. Complete surgical resection (p = 0.003), the forkhead box protein P3/CD8 (p = 0.005), and forkhead box protein P3/CD4 (p = 0.037) ratios were prognostic factors for recurrence-free survival. The CD8+ tumor-infiltrating lymphocytes rate (p = 0.037) was a prognostic factor for relapse-free survival with a threshold of 67.8/high power field. Microscopic subtype (p = 0.037) was a prognostic factor for relapse-free survival when only adenocarcinoma and squamous cell carcinoma were considered. In multivariate analysis, age (p = 0.004) and a CD8/CD4 ratio (p = 0.016) were independent predictors of overall survival. Despite the limitations of our study, our results confirm the prognostic value of tumor-infiltrating lymphocytes in non-small cell lung carcinomas and the importance of the combined quantification of their different subpopulations.

CTLA-4 Synergizes With PD1/PD-L1 in the Inhibitory Tumor Microenvironment of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is highly invasive and carries high mortality due to limited therapeutic strategies. In other solid tumors, immune checkpoint inhibitors (ICIs) target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD1), and the PD1 ligand PD-L1 has revolutionized treatment and improved outcomes. However, the relationship and clinical significance of CTLA-4 and PD-L1 expression in ICC remains to be addressed. Deciphering CTLA-4 and PD-L1 interactions in ICC enable targeted therapy for this disease. In this study, immunohistochemistry (IHC) was used to detect and quantify CTLA-4, forkhead box protein P3 (FOXP3), and PD-L1 in samples from 290 patients with ICC. The prognostic capabilities of CTLA-4, FOXP3, and PD-L1 expression in ICC were investigated with the Kaplan–Meier method. Independent risk factors related to ICC survival and recurrence were assessed by the Cox proportional hazards models. Here, we identified that CTLA-4+ lymphocyte density was elevated in ICC tumors compared with peritumoral hepatic tissues (P <.001), and patients with a high density of CTLA-4+ tumor-infiltrating lymphocytes (TILsCTLA-4 High) showed a reduced overall survival (OS) rate and increased cumulative recurrence rate compared with patients with TILsCTLA-4 Low (P <.001 and P = .024, respectively). Similarly, patients with high FOXP3+ TILs (TILsFOXP3 High) had poorer prognoses than patients with low FOXP3+ TILs (P = .021, P = .034, respectively), and the density of CTLA-4+ TILs was positively correlated with FOXP3+ TILs (Pearson r = .31, P <.001). Furthermore, patients with high PD-L1 expression in tumors (TumorPD-L1 High) and/or TILsCTLA-4 High presented worse OS and a higher recurrence rate than patients with TILsCTLA-4 LowTumorPD-L1 Low. Moreover, multiple tumors, lymph node metastasis, and high TumorPD-L1/TILsCTLA-4 were independent risk factors of cumulative recurrence and OS for patients after ICC tumor resection. Furthermore, among ICC patients, those with hepatolithiasis had a higher expression of CTLA-4 and worse OS compared with patients with HBV infection or undefined risk factors (P = .018). In conclusion, CTLA-4 is increased in TILs in ICC and has an expression profile distinct from PD1/PD-L1. TumorPD-L1/TILsCTLA-4 is a predictive factor of OS and ICC recurrence, suggesting that combined therapy targeting PD1/PD-L1 and CTLA-4 may be useful in treating patients with ICC.

Targeting CD166+ lung cancer stem cells: Molecular study using murine dendritic cell vaccine

PurposeCancer stem cells (CSC) are the most common causes of lung cancer relapse and mouse resistance to chemotherapy. CD166 was identified as CSC marker for lung cancer. Our study aimed to detect the effect of dendritic cell vaccine loaded with tumor cell lysate (TCL-DCV) on percentage of CD166+ CSC in lung of mice exposed to Benzo(a)Pyrene (BP). MethodsFemale albino mice were divided into 5 groups (22 mice per group): normal control (NC), lung cancer control (LCC) (50 mg/kg BP orally, twice weekly for four weeks), dendritic cell (DC), TCL-DCV and cisplatin. Cisplatin (6 mg/kg, intraperitoneal) was given in two doses (18th and 20th week). 1 × 106 cells of each of DC and TCL-DCV was given subcutaneously as cisplatin. At the end of experiment (22 weeks), lung tissue was used for evaluation of cytotoxic T lymphocyte antigen-4 (Ctla-4), transforming growth factor-β (Tgf-β), forkhead box protein P3 (Foxp3), programmed death ligand 1 (Pd-l1) and interleukin 12 (Il-12) gene expression using quantitative RT-PCR. The percentage of CD83+, CD8+ and CD166+ cells in lung tissue were measured using flow cytometry. ResultsThe results revealed that TCL-DCV reversed the tumorigenic effect of BP in the lung as evidenced by histopathological examination. Compared to cisplatin, dendritic cell vaccination (TCL-DCV) significantly decreased percentage of CD166+ CSC. This anticancer stemness effect was attributed to the immune-stimulatory effect as indicated by increased percentage of CD83+ and CD8+ cells, upregulation of Il-12, and downregulation of Tgf-β, Ctla-4, Pd-l1 and Foxp3 gene expression compared to LCC group. ConclusionsTCL-DCV ameliorated cancer stemness through modulating tumor immune archetypes which make it a potent therapeutic alternative to chemotherapy resistant cases.

The antioncogenic effect of Beclin-1 and FOXP3 is associated with SKP2 expression in gastric adenocarcinoma.

An overexpression of S-phase kinase-associated protein 2 (SKP2) is frequently observed in human cancer progression and metastasis, and evidence suggests that SKP2 plays a proto-oncogenic role both in vitro and in vivo. However, the function of SKP2 in gastric adenocarcinoma remains largely obscure. We investigated SKP2 expression in human gastric carcinomas.Tissue samples were acquired from 182 cases of gastric adenocarcinoma that were surgically resected from 2006 to 2012. Immunohistochemical staining for SKP2, Beclin-1, and forkhead box protein P3 (FOXP3) was performed. Pearson chi-square test was used to evaluate the associations among clinicopathological variables. The Kaplan–Meier method, the log-rank test, and the Cox proportional-hazards model were used in the analysis of the overall survival (OS) and disease-free survival (DFS).As a result, SKP2 overexpression in gastric adenocarcinomas showed a significant correlation with several favorable clinical factors, including the tumor size, T category, N category, lymphatic invasion, vascular invasion, OS, and DFS. SKP2 expression was positively correlated with the tumoral FOXP3, Beclin-1 expression, and regulatory T cell (Treg) infiltration. The difference in DFS between the SKP2 positive and negative group was attenuated by FOXP3 high expression, Beclin-1 high expression, and Tregs infiltration. Attenuation of the difference in OS by FOXP3 high expression, Beclin-1 high expression, and Tregs infiltration was not significant. In multivariable analysis, SKP2 expression was not correlated with OS and DFS.Our study showed a complex interrelationship between SKP2 and Beclin-1 and FOXP3 expression in gastric adenocarcinoma. The antioncogenic effect of Beclin-1 and FOXP3 expression in gastric adenocarcinoma is related to SKP2 expression.

Knockdown screening of chromatin binding and regulatory proteins in zebrafish identified Suz12b as a regulator of tfpia and an antithrombotic drug target

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits factor VIIa and Xa in the coagulation cascade. It has been shown that forkhead box P3 protein is a TFPI transcriptional repressor. However, there are no studies on chromatin remodeling that control TFPI expression. We hypothesized that the genome-wide knockdowns of the chromatin binding and regulatory proteins (CBRPs) in zebrafish could identify novel tfpia gene regulators. As an initial step, we selected 69 CBRP genes from the list of zebrafish thrombocyte-expressed genes. We then performed a 3-gene piggyback knockdown screen of these 69 genes, followed by quantification of tfpia mRNA levels. The results revealed that knockdown of brd7, ing2, ing3, ing4, and suz12b increased tfpia mRNA levels. The simultaneous knockdown of these 5 genes also increased tfpia mRNA levels. We also performed individual gene and simultaneous 5-gene knockdowns on the 5 genes in zebrafish larvae. We found that after laser injury, it took a longer time for the formation of the thrombus to occlude the caudal vessel compared to the control larvae. We then treated the larvae and adults with a chemical UNC6852 known to proteolytically degrade polycomb repressor complex 2, where SUZ12 is a member, and observed prolongation of time to occlude (TTO) the caudal vein after laser injury and increased tfpia mRNA levels in larvae and adults, respectively. In summary, our results have identified novel epigenetic regulators for tfpia and exploited this information to discover a drug that enhances tfpia mRNA levels and prolongation of TTO. This discovery provides the basis for testing whether UNC6852 could be used as an antithrombotic drug. This approach could be used to study the regulation of other plasma proteins, including coagulant and anticoagulant factors.

Regulatory T Cells Exhibit Interleukin-33-Dependent Migratory Behavior during Skin Barrier Disruption.

Regulatory T cells (Tregs) suppress immune responses and maintain immunological self-tolerance and homeostasis. We currently investigated relationships between skin barrier condition and Treg behavior using skin barrier-disrupted mice. Skin barrier disruption was induced by repeated topical application of 4% sodium dodecyl sulfate (SDS) on mice. The number of CD4+ forkhead box protein P3 (Foxp3)+ Tregs was higher in 4% SDS-treated skins than in controls. This increasing was correlated with the degree of acanthosis. The numbers of interleukin (IL)-10+ and transforming growth factor (TGF)-β+ Tregs also increased in 4% SDS-treated skins. Localization of IL-33 in keratinocytes shifted from nucleus to cytoplasm after skin barrier disruption. Notably, IL-33 promoted the migration of Tregs in chemotaxis assay. The skin infiltration of Tregs was cancelled in IL-33 neutralizing antibody-treated mice and IL-33 knockout mice. Thus, keratinocyte-derived IL-33 may induce Treg migration into barrier-disrupted skin to control the phase transition between healthy and inflammatory conditions.

Circulating FoxP3+ T-lymphocytes in chronic coronary artery disease: Associations with the severity of atherosclerosis and lipid metabolism

Introduction. The transcription factor forkhead box protein P3 (FoxP3) is a major regulator of T-regulatory (Treg) lymphocytes and may be expressed in T-conventional (Tconv) lymphocytes at the stage of their activation. The aim of the present study was to evaluate the quantities and features of FoxP3+ Tconv and Treg lymphocytes and their relationships with the parameters of lipid metabolism in patients with chronic coronary artery disease (CAD) depending on the severity of coronary atherosclerosis.Material and Methods. The study comprised 14 patients (8 men and 6 women) aged 66.5 ± 9.0 years with verified chronic CAD. All the patients underwent coronary angiography and assessment of atherosclerosis severity by calculation of Gensini Score index (GS). Patients were divided into the following groups: group 1 had GS &lt; 20; group 2 had GS ≥ 20. The absolute and relative counts of FoxP3+ Treg and Tconv lymphocytes and degree of FoxP3 nuclear translocation were evaluated in all patients by imaging flow cytometry. Concentrations of insulin, proprotein convertase subtilisin/kexin type 9 (PCSK9), and sortilin were assessed using enzyme-linked immunosorbent assay. Parameters of glucose metabolism and serum lipid spectrum were determined by the standard methods.Results. Counts of Treg and Tconv lymphocytes did not differ between groups of patients with different severity of atherosclerosis. However, patients with GS ≥ 20 had lower intensity of nuclear FoxP3 fluorescence in Treg and Tconv lymphocytes. GS index in the entire group of CAD patients tended to be negatively associated with the fluorescence intensity of FoxP3 in the nuclei of Treg (rs = –0.476) and Tconv lymphocytes (rs = –0.526). Multiple correlations existed between the quantitative and qualitative parameters of FoxP3+ Treg and FoxP3+ Tconv lymphocytes and metabolic parameters such as concentrations of PCSK9, sortilin, apolipoprotein B, and triglycerides/HDL cholesterol ratio.Conclusion. FoxP3 fluorescence intensity in the nuclei of T conventional lymphocytes was more sensitive marker of immunoregulatory imbalance in chronic CAD compared to counts of FoxP3+ T cells in the peripheral blood, which remained nearly unaltered with the increase in atherosclerosis severity. At the same time, markers of lipid metabolism were tightly associated with both quantitative and qualitative features of FoxP3+ T-lymphocytes.

FOXP3 facilitates the invasion and metastasis of non‑small cell lung cancer cells through regulating VEGF, EMT and the Notch1/Hes1 pathway

Forkhead box P3 (FOXP3) is a specific marker of regulatory T cells (Tregs) that is also expressed in tumour cells. Previous studies have revealed that FOXP3 can promote metastasis in several types of cancer, including non-small cell lung cancer (NSCLC); however, the underlying mechanism of FOXP3 remains unclear. The aim of the present study was to investigate the effect of FOXP3 on vascular endothelial growth factor (VEGF), epithelial-to-mesenchymal transition (EMT) and the Notch1/Hes1 pathway in NSCLC. After FOXP3 small interfering RNA (siRNAs) were transfected into A549 cells, the expression of FOXP3 mRNA and protein was determined by reverse transcription-quantitative PCR and western blotting. Cell migration and invasion were analyzed by Transwell assays. The concentrations of matrix metalloproteinase (MMP)-2, MMP-9 and VEGF in the cell supernatant were evaluated by ELISA. The expression of relevant proteins involved in EMT and Notch1/Hes1 pathway were assessed via western blotting. Additionally, the expression of FOXP3, CD31 and E-cadherin was detected by immunohistochemical (IHC) staining of 55 human NSCLC tissue samples. The results demonstrated that FOXP3 knockdown significantly inhibited the cell migratory and invasive abilities, decreased the concentrations of MMP-2, MMP-9 and VEGF, downregulated the protein expression of vimentin, N-cadherin, Notch1 and Hes family BHLH transcription factor 1 (Hes1), and upregulated the protein expression of E-cadherin. Furthermore, FOXP3 expression was positively associated with CD31+ vascular endothelial cells and negatively correlated with E-cadherin in NSCLC tissues. In addition, the Notch1/Hes1 pathway inhibitor DAPT significantly downregulated the expression of FOXP3 in a dose-dependent manner. Taken together, these findings demonstrated that FOXP3 may facilitate the invasive and migratory abilities of NSCLC cells via regulating the angiogenic factor VEGF, the EMT and the Notch1/Hes1 pathway.

Elucidating the Mechanism of Action of the Attributed Immunomodulatory Role of Eltrombopag in Primary Immune Thrombocytopenia: An In Silico Approach.

Eltrombopag is a thrombopoietin receptor (MPL) agonist approved for the treatment of primary immune thrombocytopenia (ITP). Recent evidence shows that some patients may sustain platelet counts following eltrombopag discontinuation. The systemic immunomodulatory response that resolves ITP in some patients could result from an increase in platelet mass, caused either by the direct action of eltrombopag on megakaryocytes through MPL stimulation, or potential MPL-independent actions on other cell types. To uncover the possible mechanisms of action of eltrombopag, in silico analyses were performed, including a systems biology-based approach, a therapeutic performance mapping system, and structural analyses. Through manual curation of the available bibliography, 56 key proteins were identified and integrated into the ITP interactome analysis. Mathematical models (94.92% mean accuracy) were obtained to elucidate potential MPL-dependent pathways in non-megakaryocytic cell subtypes. In addition to the effects on megakaryocytes and platelet numbers, the results were consistent with MPL-mediated effects on other cells, which could involve interferon-gamma, transforming growth factor-beta, peroxisome proliferator-activated receptor-gamma, and forkhead box protein P3 pathways. Structural analyses indicated that effects on three apoptosis-related proteins (BCL2L1, BCL2, BAX) from the Bcl-2 family may be off-target effects of eltrombopag. In conclusion, this study proposes new hypotheses regarding the immunomodulatory functions of eltrombopag in patients with ITP.

Metabolic Disturbance and Th17/Treg Imbalance Are Associated With Progression of Gingivitis.

ObjectiveThis study sought to explore the role of metabolic disturbance in immunoregulation of gingivitis targeting T helper 17 cells (Th17)/regulatory T cell (Treg).Materials and MethodsA total of 20 gingivitis patients and 19 healthy volunteers were recruited. Quantitative real time polymerase chain reaction (qRT-PCR) was used to evaluate expression patterns of Forkhead box protein P3 (Foxp3), transforming growth factor-β (TGF-β), retinoid-related orphan receptor-gammat (RORγt) and interleukin 17A (IL-17A) in the peripheral blood lymphocytes of subjects across the two groups. Moreover, the enzyme-linked immunosorbent assay (ELISA) technique was used to detect levels of TGF-β, IL-4, IL-6,TL-10 and L-17A secreted in the plasma as well as the SIgA secreted in saliva. Flow cytometry was used to detect the percentage of CD4+CD25+ Foxp3+Treg cells and the percentage of CD4+IL-17A+ Th17 cells in whole blood of subjects in both groups. Gas chromatography-mass spectrometry (GC-MS) was employed to analyze the plasma metabolites in the gingivitis patient group. Statistical analysis was applied to determine whether the plasma metabolites and related metabolic pathways significantly differed between gingivitis patients and healthy controls. Ingenuity pathway analysis (IPA) was employed to identify the potential relation between the metabolites and the Th17 and Treg related pathway.ResultsThe percentages of CD4+IL17A+Th17 cells and IL-17 significantly increased in the peripheral blood in the gingivitis group. Moreover, the upregulation of IL-17A mRNA and RORγt mRNA were also found in the gingivitis group. However, the percentage of CD4+CD25+ Foxp3+Treg cells and Foxp3 mRNA in the whole blood did not significantly change. However, TGF-β mRNA as well as TGF-β, IL-4, IL-6, IL-10 in the periperial blood and SIgA in the saliva were higher in the gingivitis group. Notably, that the ratio of Th17/Treg cells was significantly increased during peripheral circulation. Furthermore, we identified 18 different metabolites which were differentially expressed in plasma between the gingivitis and healthy control groups. Notably, the levels of cholesterol, glycerol 1-octadecanoate, d-glucose, uric acid, cyclohexaneacetic acid, 3-pyridine, tryptophan, and undecane 2,4-dimethyl were significantly up-regulated. whereas the levels of lactic acid, glycine, linoleic acid, monopalmitic acid, glycerol, palmitic acid, pyruvate, 1-(3-methylbutyl)-2,3,4,6-tetramethylbenzene, 1 5-anhydro d-altrol, and boric acid were down-regulated in the gingivitis group, relative to healthy controls. IPA showed that these metabolites are connected to IL17 signaling, TGF-B signaling, and IL10 signaling, which are related closely to Th17 and Treg pathway.ConclusionOverall, these results showed that disturbance to glycolysis as well as amino and fatty acid metabolism are associated with Th17/Treg balance in gingivitis. Impaired immunometabolism may influence some periodontally involved systemic diseases, hence it is a promising strategy in targeted development of treatment therapies.

Clonally expanded EOMES+ Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.

Expansion of GARP-Expressing CD4+CD25-FoxP3+ T Cells and SATB1 Association with Activation and Coagulation in Immune Compromised HIV-1-Infected Individuals in South Africa.

Although antiretroviral treatment lowers the burden of human immunodeficiency virus (HIV)-related disease, it does not always result in immunological recovery. This manifests as persistent chronic inflammation, immune activation or exhaustion that can promote the onset of co-morbidities. As the exact function of regulatory T (Treg) cells in HIV remains unclear, this cross-sectional study investigated three expression markers (Forkhead box protein P3 [FOXP3], glycoprotein A repetitions predominant [GARP], special AT-rich sequence binding protein 1 [SATB1]) and compared their expansion between CD4+CD25- and CD4+CD25++ T cells. Age-matched study subjects were recruited (Western Cape, South Africa) and sub-divided: HIV-negative subjects (n = 12), HIV-positive naïve treated (n = 22), HIV-positive treated based on CD4 count cells/µL (CD4 > 500 and CD4 < 500) (n = 34) and HIV-treated based on viral load (VL) copies/mL (VL < 1000 and VL > 1000) (n = 34). Markers of immune activation (CD38) and coagulation (CD142) on T cells (CD8) were assessed by flow cytometry together with FOXP3, GARP and SATB1 expression on CD4+CD25- and CD4+CD25++ T cells. Plasma levels of interleukin-10 (IL-10; anti-inflammatory marker), IL-6 (inflammatory marker) and D-dimer (coagulation marker) were assessed. This study revealed three major findings in immuno-compromised patients with virological failure (CD4 < 500; VL > 1000): (1) the expansion of the unconventional Treg cell subset (CD4+CD25-FOXP3+) is linked with disease progression markers; (2) increased GARP expression in the CD4+CD25- and CD4+CD25++ subsets; and (3) the identification of a strong link between CD4+CD25-SATB1+ cells and markers of immune activation (CD8+CD38+) and coagulation (CD8+CD142+ and D-dimer).

Gender differences in food allergy depend on the PPAR γ/NF-κB in the intestines of mice

AimsEpidemiology shows that gender affects the incidence of food allergy. However, there is a lack of evidence of gender differences in food allergies and little is known about the mechanisms. The aim of this study was to excavate potential reasons for gender differences in food allergy based on estrogen. Main methodsFemale and male BALB/c mice sensitized with ovalbumin (OVA) were established to analyze the difference in food allergy. The systemic anaphylactic, including OVA-specific IgE, OVA-specific IgG, histamine, and cytokines, was assessed using an enzyme-linked immunosorbent assay (ELISA). ELISA also detected the estradiol in serum. Western blotting and immunofluorescence were used to detect the estrogen receptor. Peroxisome proliferator-activated receptor gamma (PPARγ) implicated in immune homeostasis and nuclear factor kappa-B (NF-κB) were determined by western blotting. Immunohistochemistry and hematoxylin-eosin (H&E) staining were used to detect zonula occludens-1 (ZO-1), tryptase, forkhead box protein P3 (Foxp3), and intestinal morphology, respectively. Key findingsFemale mice were more vulnerable to food allergy. Female mice treated with OVA did exhibit more serious systemic anaphylaxis than male mice. We observed increased levels of estradiol in serum, estrogen receptor, NF-κB, and decreased levels of PPAR γ in female mice. Furthermore, the intestinal mucosal integrity and intestinal permeability were more impaired in female mice treated with OVA than male mice. SignificanceClarify the mechanism of gender differences in food allergies can provide targets in female mice and provide personalized diagnosis, management, and treatment of food allergy for female mice.

FOXP3-based immune risk model for recurrence prediction in small-cell lung cancer at stages I–III

BackgroundImmunotherapies may prolong the survival of patients with small-cell lung cancer (SCLC) to some extent. The role of forkhead box protein P3 (FOXP3) in tumor microenvironment (TME) remains controversial. We...

Cornuside alleviates experimental autoimmune encephalomyelitis by inhibiting Th17 cell infiltration into the central nervous system.

The present study was conducted to clarify the therapeutic effect of cornuside on experimental autoimmune encephalomyelitis (EAE) and its influence on T helper 17 (Th17) cell and regulatory T (Treg) cell infiltration into the central nervous system. Rats were randomly placed into four treatment groups: control, EAE, EAE+cornuside, and EAE+prednisolone. The neurological function scores of rats were assessed daily. On the second day after EAE rats began to show neurological deficit symptoms, the four groups were treated with normal saline, normal saline, cornuside (150 mg/kg), and prednisolone (5 mg/kg), respectively. The treatment was discontinued after two weeks, and the spinal cord was obtained for hematoxylin and eosin (H&E) and luxol fast blue staining, as well as retinoic acid receptor-related orphan receptor γ (RORγ) and forkhead box protein P3 (Foxp3) immunohistochemical staining. Blood was collected for Th17 and Treg cell flow cytometry testing, and the serum levels of interleukin (IL)-17A, IL-10, transforming growth factor-β (TGF-β), IL-6, IL-23, and IL-2 were measured via enzyme-linked immunosorbent assay (ELISA). Compared with rats in the EAE group, rats in the EAE+cornuside and EAE+prednisolone groups began to recover from neurological deficits earlier, and had a greater degree of improvement of symptoms. Focal inflammation, demyelination, and RORγ-positive cell infiltration were reduced by cornuside or prednisolone treatment, whereas the Foxp3-positive cell numbers were not significantly different. Meanwhile, the number of Th17 cells and the IL-17A, IL-6, and IL-23 levels were lower in the blood after cornuside or prednisolone treatment, whereas the number of Treg cells or the levels of IL-10, TGF-β, and IL-2 were not markedly different. Cornuside can alleviate symptoms of EAE neurological deficits through its anti-inflammatory and immunosuppressive effects, and Th17 cells may be one of its therapeutic targets.

Antigen-specific T cell tolerance in the prevention of post-infectious myocarditis induced with Coxsackievirus B3

Abstract Of the many types of heart disease, myocarditis is a predominant cause of heart failure in adolescents, and 10–20% of those affected can develop dilated cardiomyopathy (DCM) with infiltrates in their hearts. Autoimmunity resulting from exposure to infectious agents such as Coxsackievirus B3 (CVB) has been suggested as a possible underlying mechanism of chronic myocarditis in the development of DCM. We recently demonstrated that CVB infection could lead to the generation of pathogenic autoreactive T cells leading to a suggestion that their effector functions can be suppressed by developing antigen-specific T cell therapies. To this end, we sought to generate induced Treg cells by using biodegradable nanoparticle delivery system made of chitosan and poly(lactic-co-glycolic acid) (PLGA) entrapped with interleukin-2 and transforming growth factor termed, nanocytokines. Our data suggest that the PLGA nanocytokines can stably induce Forkhead box protein P3, a molecular marker of Treg cells accompanied with complete suppression of interferon-γ production. Our preliminary data indicate that the Treg cells induced with nanocytokines can suppress non-Treg cells in vitro leading to a suggestion that they could be used to prevent the expansion of autoreactive T cells generated in mice affected with viral myocarditis.